A non-toxic fluorogenic dye for mitochondria labeling.

BACKGROUND: Mitochondria, powerhouses of cells, are responsible for many critical cellular functions, such as cell energy metabolism, reactive oxygen species production, and apoptosis regulation. Monitoring mitochondria morphology in live cells temporally and spatially could help with the understanding of the mechanisms of mitochondrial functional regulation and the pathogenesis of mitochondria-related diseases. METHODS: A novel non-cytotoxic fluorogenic compound, AcQCy7, was developed as a mitochondria-specific dye. RESULTS: AcQCy7 emitted no fluorescent signal outside of cells, but it became fluorescent after intracellular hydrolysis of the acetyl group. The hydrolyzed fluorescent product was well retained in mitochondria, enabling long-lasting fluorescence imaging of mitochondria without cell washing. A 2-day culture study using AcQCy7 showed no sign of cytotoxicity, whereas a commonly used mitochondria-staining probe, Mitochondria Tracker Green, caused significant cell death even at a much lower concentration. Apoptosis-causing mitochondria fission was monitored clearly in real time by AcQCy7. CONCLUSIONS: A simple add-and-read mitochondria specific dye AcQCy7 has been validated in various cell models. Bright mitochondria specific fluorescent signal in treated cells lasted several days without noticeable toxicity. GENERAL SIGNIFICANCE: The probe AcQCy7 has been proofed to be a non-toxic agent for long-term mitochondria imaging.

In vivo senescence imaging nanoprobe targets the associated reactive oxygen species.

Cellular senescence, a cell-cycle arrest state upon stress or damage, can adversely impact aging and cancers. We have designed a novel near infrared fluorogenic nanoprobe, named D3, which can only be turned on by highly elevated levels of reactive oxygen species (ROS), critical players for the induction and maintenance of senescence, for real-time senescence sensing and imaging. In contrast to glowing senescent cells, non-senescent cells whose ROS levels are too low to activate the D3 signal remain optically silent. Upon systemic injection into senescent tumor-bearing mice, the D3 nanoprobe quickly accumulates in tumors, and its fluorescence signal is turned on specifically by senescence-associated ROS in the senescent tumors. The fluorescence signal at senescent tumors was 3-fold higher than that of non-senescent tumors. This groundbreaking design introduces a novel activation mechanism and a powerful imaging nanoprobe to identify and assess cellular senescence in living organisms.

Layered nanoprobe for long-lasting fluorescent cell label.

A long-lasting particle-based fluorescent label is designed for extended cell imaging studies. This onion-like nanoprobe is constructed through layer-by-layer fabrication technology. The nanoprobes are assembled with multiple layers of optically quenched polyelectrolytes, the fluorescence signal of which can be released later by intracellular proteolysis. Upon incubation with cells, the assembled nanoprobes are taken up efficiently. The tight packing and layered assembly of the quenched polyelectrolytes slow subsequent intracellular degradation, and then result in a prolonged intracellular fluorescence signal for up to 3 weeks with no noticeable toxicity.

A benzothiazole alkyne fluorescent sensor for Cu detection in living cell.

A new type of alkyne dye, 6-dimethylaminobenzothiazole alkyne (1), was developed for Cu sensing in biological system. Dye (1) offered excellent selective over a panel of ions, only Cu(I) could change the fluorescence of dye (I) by forming copper acetylide between the terminal alkyne and Cu(I). Its potential of detecting Cu in biological system was demonstrated in cell culture.

Developing a far-red fluorogenic beta-galactosidase probe for senescent cell imaging and photoablation.

A methylene blue (MB)-based beta-galactosidase (ß-gal) activatable molecule, Gal-MB, was developed for senescence imaging and light-triggered senolysis. When in contact with LacZ ß-gal or senescence-associated ß-gal (SA-ß-gal), the photoinsensitive Gal-MB becomes fluorescent. Gal-MB also offered selective phototoxicity toward LacZ ß-gal expressing cells and drug-induced senescent cells, which express SA-ß-gal, after light illumination at 665 nm.

Effective gene silencing by multilayered siRNA-coated gold nanoparticles.

Small interfering RNA (siRNA) has been widely proposed to treat various diseases by silencing genes, but its delivery remains a challenge. A well controlled assembly approach is applied to prepare a protease-assisted nanodelivery system. Protease-degradable poly-L-lysine (PLL) and siRNA are fabricated onto gold nanoparticles (AuNPs), by alternating the charged polyelectrolytes. In this study, up to 4 layers of PLL and 3 layers of siRNA (sR3P) are coated. Due to the slow degradation of PLL, the incorporated siRNA is released gradually and shows extended gene-silencing effects. Importantly, the inhibition effect in cells is found to correlate with the number of siRNA layers.

Developing visible fluorogenic 'click-on' dyes for cellular imaging.

New fluorogenic dyes were designed and synthesized based on Cu(I)-catalyzed 'click' reaction. Conjugating weakly fluorescent benzothiazole derivatives with an electron-deficient alkyne group at the 2-position with azide-containing molecules in aqueous solution form 'click-on' fluorescent adducts. Model reactions and cell culture experiment indicated that the developed 'click-on' dye could be applied to labeling various biomolecules, such as nucleic acids, proteins, and other molecules, in cells.

Near-Infrared Fluorogenic Spray for Rapid Tumor Sensing.

Surgical resection of cancerous tissues is a critical procedure for solid tumor treatment. During the operation, the surgeon mostly identifies the cancerous tissues by naked-eye visualization under white light without aid, therefore, the outcome heavily relies on the surgeon's experience. A near-infrared pH-responsive fluorogenic dye, CypH-11, was designed to be used as a sensitive cancer spray to highlight cancerous tissues during surgical operations, minimizing the surgeon's subjective judgment. CypH-11, pKa 6.0, emits almost no fluorescence at neutral pH but fluoresces brightly in an acidic environment, a ubiquitous consequence of cancer cell proliferation. After topical application, CypH-11 was absorbed quickly, and its fluorescence signal in the cancerous tissue was developed within a minute. The signal-to-background ratio was 1.3 and 1.5 at 1 and 10 min, respectively. The fluorogenic property and near-instant signal development capability enable the "spray-and-see" concept. This fast-acting CypH-11 spray could be a handy and effective tool for fluorescence-guided surgery, identifying small cancerous lesions in real time for optimal resection without systemic toxicity.

RHAMMB-mediated bifunctional nanotherapy targeting Bcl-xL and mitochondria for pancreatic neuroendocrine tumor treatment.

The incidence of pancreatic neuroendocrine tumor (PNET) has continued to rise. Due to their indolent feature, PNET patients often present with incurable, metastatic diseases. Novel therapies are urgently needed. We have previously shown that Receptor for Hyaluronic Acid-Mediated Motility isoform B (RHAMMB) and Bcl-xL are upregulated in PNETs and both of them promote PNET metastasis. Because RHAMM protein is undetectable in most adult tissues, we hypothesized that RHAMMB could be a gateway for nanomedicine delivery into PNETs. To test this, we developed a RHAMMB-targeting nanoparticle (NP). Inside this NP, we assembled small interfering RNA (siRNA) against Bcl-xL (siBcl-xL) and mitochondria-fusing peptide KLA. We demonstrated that RHAMMB-positive PNETs picked up the RHAMMB-targeting NPs. siBcl-xL or KLA alone killed only 30% of PNET cells. In contrast, a synergistic killing effect was achieved with the co-delivery of siBcl-xL and KLA peptide in vitro. Unexpectedly, siBcl-xL induced cell death before reducing Bcl-xL protein levels. The systemically injected RHAMMB-targeting NPs carrying siBcl-xL and KLA peptide significantly reduced tumor burden in mice bearing RHAMMB-positive PNETs. Together, these findings indicate that the RHAMMB-targeting nanotherapy serves as a promising drug delivery system for PNET and possibly other malignancies with upregulated RHAMMB. The combination of siBcl-xL and KLA peptide can be a therapy for PNET treatment.

Multilayered Activatable Nanoprobe for Ultra-Bright Tumor Imaging.

The development of tumor targeted probes with strong signal and high contrast is always challenging in cancer imaging. Here, a unique multilayered activatable nanoprobe (MAN) is prepared to fulfill this long-standing goal. MAN adopts a versatile layer-by-layer fabrication technique that sequentially assembles multifunctional polyelectrolytes onto nanoparticles via charge-charge interaction. Unlike the common one-probe-one-fluorochrome construct, MAN offers a dramatic fluorescence enhancement by transporting a large quantity of quenched fluorochromes for maximal signal and contrast. Excellent signal amplification and retention with negligible cytotoxicity is observed in cell study. Upon systemic injection into mice, MAN quickly accumulates in tumor and its fluorescent signal is turned on by proteases overexpressed in tumors, resulting in >700% tumor-to-normal-tissue contrast. This multilayered fabrication provides a simple and powerful universal platform to design sensitive tumor imaging probes.

Nanoparticle Delivery of miR-708 Mimetic Impairs Breast Cancer Metastasis.

Triple-negative breast cancer (TNBC) patients exhibit the worst clinical outcome due to its aggressive clinical course, higher rate of recurrence, and a conspicuous lack of FDA-approved targeted therapies. Here, we show that multilayered nanoparticles (NPs) carrying the metastasis suppressor microRNA miR-708 (miR708-NP) localize to orthotopic primary TNBC, and efficiently deliver the miR-708 cargo to reduce lung metastasis. Using a SOX2/OCT4 promoter reporter, we identified a population of miR-708low cancer cells with tumor-initiating properties, enhanced metastatic potential, and marked sensitivity to miR-708 treatment. In vivo, miR708-NP directly targeted the SOX2/OCT4-mCherry+ miR-708low tumor cells to impair metastasis. Together, our preclinical findings provide a mechanism-based antimetastatic therapeutic approach for TNBC, with a marked potential to generate miR-708 replacement therapy for high-risk TNBC patients in the clinic. To our knowledge, this gold nanoparticle-based delivery of microRNA mimetic is the first oligonucleotide-based targeted therapy for TNBC.

Total control of fat cells from adipogenesis to apoptosis using a xanthene analog.

Overcrowded adipocytes secrete excess adipokines and cytokines under stress, which results in a deregulated metabolism. This negative response to stress increases the possibility of obesity and several of its associated diseases, such as cancer and atherosclerosis. Therefore, a reduction in the number of adipocytes may be a rational strategy to relieve the undesired expansion of adipose tissue. A newly synthesized xanthene analog, MI-401, was found to have two distinct effects on the regulation of the adipocyte's life cycle. MI-401 efficiently down regulated the expression of transcription factors, PPARγ and C/EBPα, and lipogenesis proteins, FAS and FABP4. This down regulation resulted in the inhibition of adipogenesis. Without newly differentiated adipocytes, the total number of adipocytes will not increase. In addition to this inhibitory effect, MI-401 was able to actively kill mature adipocytes. It specifically triggered apoptosis in adipocytes at low micro molar concentration and spared preadipocytes and fibroblasts. These dual functionalities make MI-401 an effective agent in the regulation of the birth and death of adipocytes.

Tumor ablation using low-intensity ultrasound and sound excitable drug.

The cell membrane is a semi-fluid container that defines the boundary of cells, and provides an enclosed environment for vital biological processes. A sound excitable drug (SED) that is non-cytotoxic to cells is developed to disrupt the plasma membrane under gentle ultrasound insonation, 1MHz, 1W/cm2. The frequency and power density of insonation are within the physical therapy and medical imaging windows; thus the applied ultrasound is safe and not harmful to tissues. The insertion of SEDs into the plasma membrane is not toxic to cells; however, the intruding SEDs weaken the membrane's integrity. Under insonation, the ultrasound energy destabilized the SED disrupted membranes, resulting in membrane rupture and eventual cell death. In a xenograft breast tumor model, the SED alone or the ultrasound alone caused little adverse effects to tumor tissue, while the combined treatment triggered necrosis with a brief local insonation of 3min. The described sono-membrane rupture therapy could be a safe alternative to the currently used high-energy tissue ablation technology, which uses X-rays, gamma rays, electron beams, protons, or high-intensity focused ultrasound.

A Quick Responsive Fluorogenic pH Probe for Ovarian Tumor Imaging.

A novel cell-permeable compound, CypH-1, that is non-fluorescent at neutral pH, but fluoresces under mildly acidic conditions with a near infrared maximum emission wavelength was designed for the detection of tumors in the clinical setting. The potential of CypH-1 in ovarian cancer imaging was demonstrated using a murine model. The intraperitoneally administered CypH-1 results in a robust fluorescence signal of discrete neoplastic lesions with millimeter range resolution within few hours. Moreover, fluorescence signal is strikingly enhanced at peripheral regions of tumors at the microscopic level suggesting a sharp physiological difference at the tumor/normal tissue interface. This robust acid-activated imaging agent is expected to have significant impact in broad surgical and diagnostic applications.

Lessons learned from imaging mouse ovarian tumors: the route of probe injection makes a difference.

Patients with ovarian cancer often develop small metastatic lesions in their peritoneal cavities. Fluorescent-imaging probes that can highlight these small lesions have significant value for guiding procedures and treatment decisions. In this animal study, we demonstrated that intraperitoneal (IP) delivery of a protease-sensitive fluorescent probe resulted in the labeling of all tumors regardless of their sizes with low background signals in organs. Conversely, intravenous (IV) injections of the probe resulted in high signals in most organs and large tumors (>5 mm) but not in any of the small lesions (<2 mm).

A fluorogenic probe for ß-galactosidase activity imaging in living cells.

A cell permeable fluorescence turn-on probe, AcGQCy7, was developed to image ß-galactosidase activity in living cells. Once internalized by ß-galactosidase-expressing cells, the probe was hydrolyzed into a highly fluorescent molecule, and the fluorescent signal was retained in mitochondria for several days. This resulted in a long-lasting and strong ß-galactosidase-dependent intracellular fluorescent signal with little background fluorescence in the culture media.

p53 restoration can overcome cisplatin resistance through inhibition of Akt as well as induction of Bax.

Cisplatin (CDDP) is a chemotherapeutic agent that is widely used to treat many cancers. However, initial resistance to CDDP is a serious problem in treating cancers. In this study, in order to develop an approach to overcome resistance to CDDP, we investigated the difference in apoptotic processes between CDDP-sensitive cells and CDDP-resistant cells. By screening with CDDP sensitivity tests, we chose SNU-16 cells which are relatively resistant to CDDP, and SNU-1 cells which are sensitive to CDDP. We compared the difference between the two cell lines focusing on apoptosis. CDDP-induced reactive oxygen species (ROS) generation significantly induced loss of mitochondrial membrane potential (MMP, ∆Ψm) in SNU-1 cells, but not in SNU-16 cells. In addition, the ratio of Bax to Bcl-2 was increased by CDDP treatment in SNU-1 cells, but not in SNU-16 cells. To augment the loss of MMP, ∆Ψm in SNU-16, we inhibited Akt activity of SNU-16 cells to suppress their anti-apoptotic activity. The inhibition of Akt activity led to suppression of the anti-apoptotic protein XIAP. Akt inhibition slightly enhanced CDDP-induced apoptosis in SNU-16 cells. In addition, we enhanced pro-apoptotic activity by transfecting the cells with the wild-type p53 gene. The induction of wild-type p53 can enhance CDDP-induced apoptosis not only by inducing Bax protein but also by suppressing anti-apoptotic proteins through inhibition of Akt. In conclusion, this study suggests that the primary contributor to resistance to CDDP in SNU-16 cells may well be a failure of induction of apoptosis due to a lack of induction of pro-apoptotic proteins rather than suppression of anti-apoptotic proteins, and that restoration of p53 function can overcome the resistance to CDDP not only by augmenting the pro-apoptotic drive through p53-mediated transcriptional activation but also by inhibiting the anti-apoptotic drive through inhibition of Akt activity.

Butyrate response factor 1 enhances cisplatin sensitivity in human head and neck squamous cell carcinoma cell lines.

Cisplatin is a widely used chemotherapeutic agent in head and neck squamous cell carcinoma (HNSCC). Resistance to cisplatin is a common feature of HNSCC. To identify genes that may regulate cisplatin sensitivity, we carried out a cDNA microarray analysis of gene expression in cisplatin-sensitive and cisplatin-resistant HNSCC-derived cell lines. Among genes differentially expressed by cisplatin treatment, we have confirmed the elevated expression of butyrate responsive factor 1 (BRF1) in cisplatin-sensitive HNSCC cells and have demonstrated that the expression level of BRF1 is associated with cisplatin-sensitivity. Specific inhibition of BRF1 expression using an antisense oligodeoxynucleotide (ODN) decreased the cisplatin-sensitivity and, on the contrary, overexpression of BRF1 increased cisplatin-sensitivity in HNSCC cells. Elevated expression of BRF1 decreased the level of the human inhibitor of apoptosis protein-2 (cIAP2) and increased the caspase-3 activity in HNSCC cells. In addition, elevated expression of BRF1 decreased the expression level of enhanced green fluorescent protein (EGFP) linked to a 3' terminal AU-rich element (ARE) of cIAP2 mRNA. These findings demonstrate that BRF1 expression enhanced cisplatin sensitivity in HNSCC cells by reducing the levels of cIAP2 mRNA.

Complete genomic DNA sequence of rock bream iridovirus.

Iridovirus is a causative agent of epizootics among cultured rock bream (Oplegnathus fasciatus) in Korea. Here, we report the complete genomic sequence of rock bream iridovirus (RBIV). The genome of RBIV was 112080 bp long and contained at least 118 putative open reading frames (ORFs), and its genome organization was similar to that of infectious spleen and kidney necrosis virus (ISKNV). Of the RBIV's 118 ORFs, 85 ORFs showed 60-99% amino acid identity to those of ISKNV. Phylogenetic analysis of major capsid protein (MCP), DNA repair protein RAD2, and DNA polymerase type-B family indicated that RBIV is closely related to red sea bream iridovirus (RSIV), Grouper sleepy disease iridovirus (GSDIV), Dwarf gourami iridovirus (DGIV), and ISKNV. The genome sequence provides useful information concerning the evolution and divergence of iridoviruses in cultured fish.